Korean J Physiol Pharmacol.  2023 Sep;27(5):437-448. 10.4196/kjpp.2023.27.5.437.

Paeonol accelerates skin wound healing by regulating macrophage polarization and inflammation in diabetic rats

• Zhang Z
• Chen T
• Liu W
• Xiong J
• Jiang L
• Liu M

• KMID: 2545534
• DOI: http://doi.org/10.4196/kjpp.2023.27.5.437

Abstract

Diabetic ulcer is usually seen in people with uncontrolled blood sugar. Reportedly, many factors such as impaired glucose metabolism, and macrovascular and microvascular diseases caused angiogenesis disorders and delayed the healing of diabetic ulcers, thus affecting the body's metabolism, nutrition, and immune function. This study aimed to explore the effect of paeonol on skin wound healing in diabetic rats and the related mechanism. A rat model of diabetic ulcer was established. High glucose-treated mouse skin fibroblasts were co-cultured with M1 or M2-polarized macrophages treated with or without paeonol. H&E and Masson staining were used to reveal inflammatory cell infiltration and collagen deposition, respectively. Immunohistochemistry visualized the expression of Ki67, CD31, and vascular endothelial growth factor (VEGF). Western blot was used to detect interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-4, IL-10, CD31, VEGFA, and collagen I/III. The expression of iNOS and arginase 1 was revealed by immunofluorescence staining. Paeonol treatment augmented collagen deposition and the expression of Ki67, CD31, VEGF, and macrophage M2 polarization markers (IL-4 and IL-10) and reduced wound area, inflammatory cell infiltration, and macrophage M1 polarization markers (IL-1β and TNF-α) in the ulcerated area. In vitro, paeonol treatment promoted M2-polarization and repressed M1-polarization in macrophages, thereby improving the repair of cell damage induced by high glucose. Paeonol accelerates the healing of diabetic ulcers by promoting M2 macrophage polarization and inhibiting M1 macrophage polarization.

Keyword

Diabetes; Macrophage polarization; Paeonol; Wound healing

Figure

• 

  Fig. 1 Paeonol promotes wound healing in diabetic rats. The wound healing (A), wound area (B), and wound closure rate (C) of diabetic ulcers before and after paeonol treatment. *p < 0.05, compared with the control group. #p < 0.05, compared with the DM group. DM, diabetes mellitus.

• 

  Fig. 2 Paeonol reduces inflammation and promotes collagen deposition in diabetic ulcers. Ulcerated DM rats were treated with or without paeonol. (A) H&E assessment of the pathological characteristics of the ulcers. (B) Masson staining for revealing collagen deposition in the ulcers. Immunohistochemical detection of the expression of Ki67 (C), CD31 and VEGF (D). The data were expressed as mean ± standard deviation. Each group had 5 rats. Scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, ###p < 0.001, compared with the DM group. DM, diabetes mellitus; VEGF, vascular endothelial growth factor; IHC, immunohistochemistry.

• 

  Fig. 3 Paeonol promotes M2 macrophage polarization in diabetic ulcers. Ulcerated DM rats were treated with or without paeonol. (A, B) Western blot was used to detect the expression of inflammatory factors IL-1β, TNF-α, IL-4 and IL-10 and macrophage markers CD86 and CD206 in the ulcers on the 21st day. (C, D) Immunofluorescence was used to detect the expression of iNOS and arginase 1 in the ulcers. Scale bar = 50 μm. The data were expressed as mean ± standard deviation. Each group had 5 rats. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, ##p < 0.01, compared with the DM group. DM, diabetes mellitus; IL, interleukin; TNF-α, tumor necrosis factor α.

• 

  Fig. 4 Paeonol improves the repair of high glucose-induced cell damage by modulating macrophage polarization. (A) MTS kit was used to assess the effect of paeonol treatment (0, 5, 10, 25, and 50 μM) on macrophage viability. RAW264.7 cells were polarized into M1 and M2 macrophages under LPS + IFN-γ treatment and IL-4 treatment, respectively. Then M1 or M2 macrophages were treated with paeonol and RT-qPCR (B) and immunofluorescence (C) was used to detect the expression of the M1 marker iNOS and the M2 marker arginase 1. Next, HG-treated mouse skin fibroblasts were co-cultured with M1 or M2-polarized macrophages treated with or without paeonol. (D, E) Western blot was used to detect the expression of inflammatory factors (IL-1β, TNF-α, IL-4, and IL-10), angiogenic factors (CD31 and VEGFA), and collagen I/III in the supernatant of mouse skin fibroblasts. Scale bar = 50 μm. The data were presented as mean ± standard deviation of three independent replicate experiments. *p < 0.05, compared with the control group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group. #p < 0.05, compared with the HG, LPS + IFN-γ, or LPS + IL-4 group. &p < 0.05, &&p < 0.01, &&&p < 0.001, compared with the HG + M1 or HG + M2 group. HG, high glucose; LPS, lipopolysaccharide; IFN, interferon; IL, interleukin; TNF-α, tumor necrosis factor α; RT-qPCR, Reverse transcription-quantitative polymerase chain reaction.

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